J. Org. Chem. 2000, 65, 8465-8469
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‘Upenamide: An Unprecedented Macrocyclic Alkaloid from the Indonesian Sponge Echinochalina sp. Jorge I. Jime´nez,† Gilles Goetz,† Christina M. S. Mau,† Wesley Y. Yoshida,† Paul J. Scheuer,*,† R. Thomas Williamson,‡ and Michelle Kelly§ Department of Chemistry, University of Hawai’i at Manoa, Honolulu, Hawaii 96822-2275, College of Pharmacy, Oregon State University, Corvallis, Oregon 97331, and Marine Ecology and Aquaculture Group, National Institute of Water and Atmospheric Research (NIWA), Taihoro Nukurangi, Private Bag 109-695 Newmarket, Auckland, New Zealand Received May 23, 2000
‘Upenamide (1) represents a new class of macrocyclic marine alkaloid possessing both spirooxaquinolizidinone and hemiaminal ring systems. It was isolated from the Indonesian sponge Echinochalina sp. The gross structure of 1 was elucidated by spectroscopic methods and accurate mass measurements. A suggestion is made as to its biogenetic origin. Over the past 15 years, an increasing number of macrocyclic diamine alkaloids have been reported from marine sponges. To date, more than 10 classes of polycyclic alkaloids have been described in the literature: saraines (from Reniera sarai, family Chalinidae),1 haliclamines (from Haliclona sp., family Chalinidae),2 xestospongins (from Xestospongia exigua, family Petrosiidae),3 petrosins (from Petrosia seriata, family Petrosiidae),4 papuamines (Haliclona sp., family Chalinidae),5 manzamines ()keramamine, from Xestospongia sp., family Petrosiidae, and Pellina sp., family Oceanapiidae),6 cyclostelletamines (from Stelleta maxima),7 mandangamine (from Xestospongia ingens, family Petrosiidae),8 xestocyclamines (from Xestospongia sp., family Petrosiidae),9 ircinols (Ircina sp. and Amphimedon sp.),10 halicyclamine A (from Haliclona sp., family Chalinidae),11 ingenamines (from Xestospongia ingens, family Petrosiidae),12 and * To whom correspondence should be addressed: Tel: (808)-9565904. Fax: (808)-956-5908. E-mail:
[email protected]. † University of Hawai’i at Manoa. ‡ Oregon State University. § National Institute of Water and Atmospheric Research. (1) (a) Cimino, G.; De Stefano, S.; Scognamiglio, G.; Sodano, G.; Trivellone, E. Bull. Soc. Chim. Belg. 1986, 95, 783-800. (b) Cimino, G.; Mattia, C. A.; Mazzarella, L.; Puliti, R.; Scognamiglio, G.; Spinella, A.; Trivellone, E. Tetrahedron 1989, 45, 3863-3972. (c) Cimino, G.; Scognamiglio, G.; Spinella, A.; Trivellone, E. J. Nat. Prod. 1990, 53, 1519-1525. (d) Guo, Y.-W.; Madaio, A.; Scognamiglio, G.; Trivellone, E.; Cimino, G. Tetrahedron 1996, 52, 8341-8348. (e) Guo, Y.-W.; Madaio, A.; Trivellone, E.; Scognamiglio, G.; Cimino, G. Tetrahedron 1996, 52, 14961-14974. (f) Guo, Y.-W.; Trivellone, E.; Scognamiglio, G.; Cimino, G. Tetrahedron Lett. 1998, 39, 463-466. (2) Fusetani, N.; Yasumoro, K.; Hirota, H. Tetrahedron Lett. 1989, 30, 6891-6894. (3) (a) Nakagawa, M.; Endo, M.; Tanaka, N.; Gen-Pei, L. Tetrahedron Lett. 1984, 25, 3227-3230. (b) Kobayashi, M.; Miyamoto, Y.; Kitagawa, I. Chem. Pharm. Bull. 1989, 37, 1676. (c) Quirion, J.-C.; Sevenet, T.; Husson, H.-P.; Weniger, B.; Debitus, C. J. Nat. Prod. 1992, 55, 1505-1508. (d) Venkateswarlu, Y.; Venkata Rami Reddy, M.; Venkataswara Rao, J. J. Nat. Prod. 1994, 57, 1283-1285. (e) Venkata Rami Reddy, M.; Faulkner, D. J. Nat. Prod. Lett. 1997, 11, 53-59. (4) (a) Breakman, J. C.; Daloze, D.; Macedo de Abreu, P.; PicciniLeopardi, C.; Germain, G.; Meerssche, M. Tetrahedron Lett. 1982, 23, 4277-4281. (b) Breakman, J. C.; Daloze, D.; Defay, N.; Zimmerman, D. Bull. Soc. Chim. Belg. 1984, 93, 941-944. (c) Breakman, J. C.; Daloze, D.; Cimino, G.; Trivellone, E. Bull. Soc. Chim. Belg. 1988, 97, 519-524. (d) Kobayashi, M.; Kawazoe, K.; Kitagawa, I. Tetrahedron Lett. 1989, 30, 4149-4152. (5) (a) Baker, B. J.; Scheuer, P. J.; Shoolery, J. N. J. Am. Chem. Soc. 1988, 110, 965-966. (b) Fahy, E.; Molinski, T. F.; Harper, M. K.; Sullivan, B. W.; Faulkner, D. J. Tetrahedron Lett. 1988, 29, 34273428.
halitoxins (from Haliclona rubens, family Chalinidae, and Callyspongia fibrosa, family Callyspongiidae).13 Despite possessing quite different structural frameworks, they appear to be biogenetically derived from bis-3-alkylpyridine or reduced bis-3-alkylpyridine units.14 In our continuing search for new biologically active marine natural products from the Indo-Pacific area, work was begun on the crude extract of a tough and elastic, reddish brown, branching sponge, Echinochalina sp. (Protolithospongia) (order Poecilosclerida, family Microcionidae), collected from Derawan Island, Indonesia. The freeze-dried sponge was extracted in methanol and dichloromethane, and the crude extract was subjected to liquid-liquid partition (Kupchan procedure)15 followed by size-exclusion, normal, and reversed-phase chroma(6) (a) Sakai, R.; Higa, T.; Jefford, C. W.; Benardinelli, G. J. J. Am. Chem. Soc. 1986, 108, 6404-6405. (b) Sakai, R.; Kohmoto, S.; Higa, T.; Jefford, C. W.; Benardinelli, G. J. Tetrahedron Lett. 1987, 28, 54935496. (c) Nakamura, H.; Deng, S.; Kobayashi, J.; Ohizumi, Y.; Tomataka, Y.; Matsuzaki, T. Tetrahedron Lett. 1987, 28, 621-624. (d) Ichiba, T.; Sakai, R.; Kohmoto, S.; Sancy, G.; Higa, T. Tetrahedron Lett. 1988, 29, 3083-3086. (e) Ichiba, T.; Corgiat, J. M.; Scheuer, P. J.; Borges, M. K. J. Nat. Prod. 1994, 57, 168-170. (f) Tsuda, M.; Kawasaki, N.; Kobayashi, J. Tetrahedron Lett. 1994, 35, 4387-4388. (g) Kobayashi, M.; Chen, Y.-J. Aoki, S.; In, Y.; Ishida, T.; Kitagawa, I. Tetrahedron Lett. 1995, 51, 3727-3736. (h) Ohtani, I. I.; Ichiba, T.; Isobe, M.; KellyBorges, M.; Scheuer, P. J. J. Am. Chem. Soc. 1995, 117, 10743-10744. (7) Fusetani, N.; Asai, N.; Matsunaga, S.; Honda, K.; Yasumuro, K. Tetrahedron Lett. 1994, 35, 3967-3970. (8) Kong, F.; Andersen, R. J.; Allen, T. M. J. Am. Chem. Soc. 1994, 116, 6007-6008. (9) (a) Rodriguez, J. M.; Peters, B.; Kurz, L.; Schatzman, R.; McCarley, D.; Lou, L.; Crews, P. J. Am. Chem. Soc. 1993, 115, 1043610437. (a) Rodriguez, J. M.; Crews, P. Tetrahedron Lett. 1994, 35, 4719-4722. (10) (a) Tsuda, M.; Kawasaki, N.; Kobayashi, J. Tetrahedron 1994, 50, 7957-7960. (b) Kondo, K.; Shigemori, H.; Kikachi, Y.; Ishibashi, M.; Sasaki, T.; Kobayashi, J. J. Org. Chem. 1992, 57, 2480-2483. (c) Kobayashi, J.; Tsuda, M.; Kawasaki, N.; Matsumoto, K.; Adachi, T. Tetrahedron Lett. 1994, 35, 4383-4386. (11) (a) Jaspars, M.; Pasupathy, V.; Crews, P. J. Org. Chem. 1994, 59, 3253-3255. (12) (a) Kong, F.; Andersen, R. J.; Allen, T. M. Tetrahedron 1994, 35, 1643-1646. (b) Kong, F.; Andersen, R. J.; Allen, T. M. Tetrahedron 1994, 50, 6137-6144. (c) Kong, F.; Andersen, R. J. Tetrahedron 1995, 51, 2895-2906. (13) (a) Schmitz, F.; Hollenbeak, K. H.; Campbell, D. C. J. Org. Chem. 1978, 43, 3916-3922. (b) Talpir, R.; Rudi, A.; Ilan, M.; Kashman, Y. Tetrahedron Lett. 1992, 33, 3033-3034. (c) Davies-Coleman, M. T.; Faulkner, D. J.; Dubowchik, G. M.; Roth, G. P.; Polson, C.; Fairchild, C. J. Org. Chem. 1993, 58, 5925-5930. (14) Baldwin, J. E.; Whitehead, R. C. Tetrahedron Lett. 1992, 33, 2059-2062.
10.1021/jo000789w CCC: $19.00 © 2000 American Chemical Society Published on Web 11/17/2000
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Figure 1. Proposed biogenesis of ‘upenamide (1).
tography to afford ‘upenamide (1, Figure 1).16 ‘Upenamide represents a new class of macrocyclic diamine alkaloid possessing both spirooxaquinolizidinone and hemiaminal ring systems. ‘Upenamide (1) was obtained as an amorphous white solid with a molecular formula of C32H46N2O4 as established by HRFABMS, m/z [M + H]+ 523.3538. The 13C NMR spectrum of 1, which showed resolved resonances for all 32 carbon atoms (2 C, 15 CH, 15 CH2) (Table 1), contained nine deshielded resonances that could be assigned to olefinic and amide carbonyl carbons. IR bands at 3411 and 1676 cm-1 and 13C NMR resonances at δ 70.0 (CHOH: C-11) and δ 169.4 (CO: C-4) were assigned to a secondary alcohol and an amide carbonyl unit. The anchor points for structural analysis were the well-resolved 1H NMR double bond resonances between δ 6.72 and δ 5.48. The 1H-1H COSY and HOHAHA NMR spectra justified the connectivities between C-11/C-14 and between C-16/C-22, respectively, with a cis double bond geometry at C-12/C-13 and all-trans double bond geometry at C-16/C-21 based on proton coupling constants (Chart 1). Fragments I and II were connected through C-15 (δ 44.8) on the basis of HMBC correlation between H-15 (δ 2.64) and C-16 (δ 135.8) as well as vicinal coupling (COSY spectrum) between H-15 and H-16 (δ 5.72). Furthermore, fragment III could also be connected to fragments I/II through C-9 (δ 44.3) on the basis of HMBC correlations between C-9 and H-11 (δ 4.82)/H-15. The proton resonance at δ 4.18 (Heq-6; nitrogen-bearing methylene) was instrumental in completing the structure of fragment III. Despite overlapping signals, the 1H-1H COSY spectrum showed cross-peaks between Heq-6 and Hax-6 (δ 2.88), H2-7 (δ 1.52, 2.08), and H2-8 (δ1.61, 1.99). The C-6 (δ (15) Kupchan, S. M.; Britton, R. W.; Ziegler, M. F.; Sigel, C. W. J. Org. Chem. 1973, 38, 179-179. (16) The name is coined from ‘upena, fishing net or trap in Hawaiian, which reflects the meshlike structure of the compound.
Chart 1.
Structural Fragments Deduced from the COSY and HMBC Spectrum
1H-1H
41.9)/C-7 (δ 21.7)/C-8 (δ 22.2) connection was also confirmed by HMBC correlations from Hax-6 to C-4/C-8/C10 (δ 88.7). Connection of fragments I/II/III completed rings B and C and the point of attachment of the alkenyl side chain at C-15. The proton resonances at δ 4.78 (H-10; nitrogen- and oxygen-bearing methine) and δ 3.62 (H-2; oxygen-bearing methine) were pivotal in structure analysis/connection of fragments IV to V. The proton signal associated with H-10 showed no COSY cross-peaks to any other proton, but the HMBC spectrum showed correlations between H-10 and C-2 (δ 73.3)/C-4/C-9/C-11/C-15 linking these two fragments. In contrast, the proton signal at δ 3.62 showed COSY cross-peaks to H-3 (δ 2.27, 2.38)/H-36b (δ 1.43), and no HMBC correlations were observed. These observations completed the structure of ring A, the point of attachment of the C-33/C-36 side chain at C-2, and the connection of ring A to the B/C ring system. Thus, the lower portion of ‘upenamide consists of a novel spirooxaquinolizidinone unit.
‘Upenamide: An Unprecedented Macrocyclic Alkaloid
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Table 1. NMR Spectral Data for ‘Upenamide (1) with COSY, NOESY, and HMBCa 1H
position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 a
(mult; J ) Hz)
3.62 (ddt; 3, 3, 11) eq: 2.27 (dd; 3, 17) ax: 2.38 (dd; 11, 17)
13C
73.3 39.6
COSY
NOESY
HMBC
3eq, 3ax, 36 2, 3ax 2, 3eq
10, 35, 36 2, 3ax, 36 3eq, 36
6ax, 7 6eq, 7
6ax, 7 10
4, 8, 10
8ax 7, 8eq
10
6, 7, 9, 10, 11 6, 7, 9, 11
2, 6ax, 8ax, 16, 17 12, 15 11, 13, 14 12, 14
2, 4, 9, 11, 15 9, 10 9, 11, 13, 14 14 9, 12, 13 9 8, 9, 11, 14, 16, 17 9, 14, 18 15, 18, 19 16, 17, 20 17, 20, 21 22 19 20, 21, 32 21
4 2, 4, 36
169.4 eq: ax: eq: ax: eq: ax:
4.18 (ddd; 5, 5, 15) 2.88 (ddd; 5, 10, 13) 2.08 (m) 1.52 (m) 1.99 (m) 1.61 (m)
4.78 (s) 4.82 (s) 5.48 (dd; 1.1, 10) 5.63 (ddd; 2, 2, 10) eq: 2.00 (m) ax: 1.90 (m) 2.64 (ddd; 6, 11, 11) 5.72 (dd; 11, 15) 6.64 (dd; 11, 15) 6.01 (dd; 11, 15) 5.97 (dd; 11, 15) 6.72 (dd, 11, 15) 5.69 (ddd; 4, 11, 15) a: 3.58 (t; 11) b: 3.16 (m) a: 2.77 (ddd; 3, 12, 12) b: 2.57 (br d; 12) a: 1.71 (m) b: 1.66 (m) a: 1.76 (m) b: 1.30 (m) 1.63 (m) a: 1.72 (m) b: 1.59 (m) a: 1.36 (m) b: 1.28 (m) 3.17 (m) 4.12 (s) a: 1.51 (m) b: 1.46 (m) a: 1.68 (m) b: 1.53 (m) a: 1.68 (m) b: 1.53 (m) a: 1.65 (m) b: 1.43 (m)
41.9 21.7 22.2 44.3 88.7 70.0 133.1 126.6 30.8
12, 13 11, 13, 14 11, 12, 14
44.8 135.8 129.3 130.6 128.8 131.3 130.2 56.4
14, 16 15, 17 16, 18 17 20 19, 20, 22 20, 22a, 22b 21, 22b 21, 22a
49.0
24b, 25a, 25b 24a, 25a, 25b
11 10, 15, 17 10, 14 16 21 22, 32 19, 22, 32 32
25.9 23.9 35.7 29.8
32
29, 32
76.3
29, 33
28, 32, 33
86.3 34.8
27
21, 22, 25, 27, 30
28.1
22, 26, 28, 30 34, 35
24.21 21.18 34.5
34, 35 2, 34, 35
Spectra of 1 were recorded in CD3OD at 500 MHz for 1H NMR and 125 MHz for
Completion of fragment VI was accomplished by HMBC and 1H-1H COSY correlations. The proton signal resonating at δ 4.12 (H-32, nitrogen- and oxygen-bearing methine) indicated only one 1H-1H COSY correlation to H-27 (δ 1.63); HMBC cross-peaks to C-22 (δ 56.4)/C-30 (δ 76.3) established the connections between C-32 (δ 86.3) and C-22 (across N-23) and C-30 (across O-31), thus providing the point of attachment of the alkenyl chain to ring D. The diagnostic proton signals of H2-24 (δ 2.57, 2.77) showed COSY cross-peaks to H2-25 (δ 1.66, 1.71), whereas H-30 (δ 3.17) showed COSY correlations to H229 (δ 1.28, 1.36) and H2-33 (δ 1.46, 1.51), providing sufficient evidence to complete assignment of rings D/E and also the point of attachment of the C-33/C-36 chain. Connection of all fragments yielded ‘upenamide (1), containing a unique hemiaminal ring system in the upper portion of the molecule. Compound 1 showed no Bohlman absorptions in its IR spectrum, thus suggesting absence of a trans-oxaquinolizidine system.17 The NOESY spectrum of 1 indicated
13C
NMR.
cross-peaks between H-10 and H-2/H-6ax/H-8ax/H-16/H17, clearly indicating that rings A/B are cis-oriented and the C-15 carbon on ring C is β-oriented with reference to rings A and B. Further evidence was obtained from the 1H and 13C NMR data of 1 as compared to previously isolated oxaquinolizidine-containing compounds.3 Particularly interesting is the downfield chemical shift for the H-10 signal (δ 4.78) due to the effects of the lone pair electrons of N-4,18 and an opposite upfield shift is observed for C-10 (δ 88.7).19 Also, the NOESY spectrum of 1 revealed a cross-peak between H-11 and H-15, thus (17) (a) Bohlman, F. Angew. Chem. 1957, 69, 641-643. (b) Uskokovic, M.; Bruderer, H.; von Planta, C.; Williams, T.; Brossi, A. J. Am. Chem. Soc. 1964, 86, 3364-3367. (c) Crabb, T. A.; Newton, R. F.; Jackson, D. Chem. Rev. 1971, 71, 109-126. (18) (a) Rosen, W. E. Tetrahedron Lett. 1961, 2, 481-484. (b) Rosen, W. E.; Shoolery, J. N. J. Am. Chem. Soc. 1961, 83, 4816-4819. (19) In xestospongin C, which contains both cis and trans oxaquinolizidine systems, the proton signal for H-10 appears at δ 4.40 in the cis case versus H-10′, which appears at δ 3.13 in the trans case. The 13C signal for C-10 appears at δ 88.18 in the cis case versus C-10′, which appears at δ 96.22 in the trans case.3a
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addition of C-9 onto C-15 initiated by the nitrogen lone pair (Figure 1). In a purely formal sense, ‘upenamide may be considered to be made up of two NCO fragments connected by two alkyl chains. Isolation of 1 expands our knowledge of the biosysnthetic pathways leading to 3-alkylpyridine and bis-3-alkylpyridine. In bioassays 1 does not show in vitro growth inhibition effects against P388, A549, and HT29 cancer cell lines. Figure 2. Relevant NOEs obtained from 1D and 2D NOE spectra.
suggesting that both C-11 hydroxyl and C-15 alkenyl chain are R-oriented with respect to ring C (Figure 2). The absolute configuration for the lower portion of ‘upenamide was determined through Mosher analysis.20,21 Chemical shift differences between the S- and R-Mosher esters22 suggested the 11R configuration for the carbinol center and the absolute configuration for all stereogenic centers on rings A/B/C as 2R,9S,10S,11R,15R (Figure 3). The relative configuration of the hemiaminal portion of ‘upenamide was not straightforward, due to the severe crowding of upfield resonances between δ 1.2 and δ 1.8; however, tracing the NOESY and 1D gNOE NMR23 spectra between H-32 to H-27 and H-32 to H-30 provided the final piece of the puzzle: a cis decalin-like arrangement of the D/E rings (Figure 2). A cis-junction of rings D/E is also confirmed, because H-27 and H-32 appear as broad singlets in the proton spectrum, attesting to a very small coupling constant and lowfield displacement of the anomeric proton H-32 (δ 4.12). Syntheses of rare mixed O-N-bisheterobicycles with cis-junction selectivity have been conducted by Duhamel and co-workers, which clearly supports the relative configuration of the hemiaminal portion of ‘upenamide.24 To determine the absolute configuration of rings D/E, 1 was treated with NaCNBH3 in THF:MeOH (1:1) under reflux to afford the corresponding piperidine diol 2.25 Unfortunately, no chemical shift differences were seen upon derivatization of 2 to its corresponding S- and R-Mosher esters, presumably due to the lack of proper conformation. Attempts to use more exotic derivatizing reagents were precluded because of the lack of sample. ‘Upenamide (1) has a skeleton without prior precedent and appears to be closely related to a hypothetical haliclamine such as 4.2,14,26 The basic biogenetic building blocks of 1 consist of ammonia, a three carbon unit present in propenal, and a variable saturated or unsaturated linear dialdehyde accounting for the formation of the bis-3-alkyldiyhydropyridine macrocycle 4. Formation of the C ring in 1 could be envisaged as a Michael-type (20) Ohtani, I,; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem. Soc. 1991, 113, 4092-4096. (21) S- and R-MTPA esters were prepared by reacting 1 with R-MTPA-Cl or S-MTPA-Cl in dry pyridine at room temperature. Attempts to purify either Mosher ester led to complete decomposition and loss of material. After careful examination of the 1H NMR spectra of both esters, some important information could be extracted for chemical shift comparison. (22) S-MTPA ester of 1: 1H NMR (500 MHz, pyridine-d5) δ 4.598 (H-10), 6.24 (H-11), 5.455 (H-12), 5.841 (H-13), 5.879 (H-16), 6.716 (H17), 6.00 (H-18), 6.161 (H-19), 6.936 (H-20), 5.643 (H-21). R-MTPA ester of 1: 1H NMR (500 MHz, pyridine-d5) δ 4.653 (H-10), 6.3125 (H-11), 5.2075 (H-12), 5.756 (H-13), 5.874 (H-16), 6.732 (H-17), 6.03 (H-18), 6.168 (H-19), 6.9505 (H-20), 5.639 (H-21). (23) Stott, K.; Keeler, J.; Van, Q. N.; Shaka, A. J. J. Magn. Reson. 1997, 125, 302-324. (24) Duhamel, P.; Deyine, A.; Dujardin, G.; Ple´, G.; Poirier, J.-M. J. Chem. Soc., Perkin Trans. 1, 1995, 2103-2114.
Experimental Section Spectral Analysis. NMR spectra were determined on a General Electric GN Omega 500 spectrometer operating at 500 MHz for 1H and 125 MHz for 13C. Gradient NOE experiments were carried on a Bruker DRX600. 1H chemical shifts are referred to CD3OD (offset set to δ 3.30 for CHD2OD impurity); 13 C chemical shifts are referred to CD3OD (49.0 ppm). Homonuclear 1H connectivities were determined by using the 2D double-quantum filtered COSY and 1D decoupling experiments. Homonuclear 1H NOEs were obtained by difference NOE experiments using a 2 s irradiation period. One-bond heteronuclear 1H-13C connectivities were determined by 2D proton-detected HMQC experiments; two- and three-bond 1H13C connectivities were determined by 2D proton-detected HMBC experiments. High-resolution mass spectra were determined in the FAB modes. Optical rotations were measured on a Jasco-DIP-700 instrument using methanol at 20 °C at the sodium D line (589 nm). IR spectra were recorded on a Perkin-Elmer 1600 FTIR. Animal Material. The sponge was collected in March 1996 at a depth of 80 feet at Old Pier, Derawan, Indonesia (2° 17'’ 28” N, 118° 14’ 13” E). The sponge is erect, branching to produce-fan-shape blades, and has a smooth, velvety upper surface. The opposing face is covered in small holes arranged in a circle. The texture is very firm and fibrous, with a central diffuse axis. In life the sponge is reddish brown, and light brown in ethanol. The skeleton is composed of robust sponginbound reticulate tracts, heavily cored by oxea that run parallel to the axis of the sponge; each tract is echinated heavily by smaller oxea. At the surface, oxea form dendritic diverging tracts. The sample is an undescribed species of the genus Echinochalina (Protolithospongia, Order Poecilosclerida, Family Microcionidae). A voucher specimen has been deposited at the Natural History Museum, London, United Kingdom (BMNH 1996.11.20.2). Extraction and Isolation. The wet sponge (588 g) was extracted in methanol (1.5 L) and methylene chloride (0.5 L). The solutions were combined and concentrated to dryness. The crude extract was partitioned between chloroform/water (1:1) and the aqueous layer reextracted with n-butanol to afford fractions A (63 mg), B (170 mg), and C (214 mg). Fractions A and B were combined and subjected to liquid-liquid partitioning using the Kupchan procedure.15 The crude material from (25) ‘Upenamide (1) (2.5 mg) was refluxed in THF:MeOH (1.0 mL, 1:1, 2 h) and the crude product purified by reversed-phase HPLC to yield 1.5 mg of the piperidine diol 2: 1H NMR (500 ΜΗz, CDCl3) δ 3.68 (m, H-2), 2.46 (dd, J ) 3 and 17 Hz, H-3eq), 2.26 (dd, J ) 12 and 17 Hz, H-3ax), 4.45 (dt, J ) 4, 4, and 14 Hz, H-4eq), 2.71 (ddd, J ) 4, 13, and 13 Hz, H-4ax), 2.16 (m, H-7eq), 1.59 (m, H-7ax), 4.66 (s, H-10), 4.99 (s, H-11), 5.53 (dd, J ) 1.5 and 10 Hz, H-12), 5.63 (m, H-13), 2.051.89 (m, H2-14), 2.65 (ddd, J ) 6, 11, and 11 Hz, H-15), 5.65 (dd, J ) 11 and 15 Hz, H-16), 6.53 (t, J ) 11 Hz, H-17), 5.95 (dd, J ) 11 and 15 Hz, H-18), 5.93 (dd, J ) 11 and 15 Hz, H-19), 6.55 (t, J ) 11 Hz, H-20), 5.77 (ddd, J ) 4, 11, and 15 Hz, H-21), 3.34 (dd, J ) 3 and 112 Hz, H-22a), 2.56 (dd, J ) 12 and 12 Hz, H-22b), 2.77 (m, H-24a), 2.04 (m, H-24b), 1.90 (m, H-27), 3.71 (q, J ) 4.5 Hz, H-30), 3.08 (m, H-32a), 1.34 (m, H-32b), 1.65 (m, H-36a), 1.61 (m, H-36b). Severe overlapping obscured signals for H-8, H-25, H-26, H-28, H-29, and H-33 to H-35. 13C NMR (125 MHz, CDCl ) δ 72.7 (C-2), 38.4 (C-3), 167.8 (C-4), 40.7 3 (C-6), 21.1 (C-7), 42.4 (C-9), 70.7 (C-11), 130.7 (C-12), 125.6 (C-13), 30.3 (C-14), 42.6 (C-15), 133.7 (C-16), 127.9 (C-17), 128.5 (C-18), 128.6 (C19), 128.0 (C-20), 132.2 (C-21), 61.8 (C-22), 56.2 (C-24), 30.1 (C-27), 70.9 (C-30), 58.4 (C-32), 34.8 (C-36). (26) Andersen, R. J.; Van Soest, R. W. M.; Kong, F. In Alkaloids: Chemical and Biological Perspectives; Pelletier, W. S., Ed., Pergamon: New York, 1996; Vol. 10, Chapter 3, pp 301-355.
‘Upenamide: An Unprecedented Macrocyclic Alkaloid
J. Org. Chem., Vol. 65, No. 25, 2000 8469
Figure 3. ∆δ values obtained for MTPA esters of 1. the methylene chloride extraction was loaded onto an ODS flash column eluting with various mixtures of methanol:water to methanol affording seven major fractions (A-G). Fraction E was further separated by Sephadex LH-20 [methanol/ chloroform (1:1)], normal phase column chromatography (hexanes/ethyl acetate), and reversed-phase HPLC (Cosmosil 5 C18-AR, 80:20 MeCN/water) to yield ‘upenamide (2.2 mg). Using the same isolation protocol, a freeze-dried sponge sample (100.0 g) afforded additional compound 1 (5.0 mg). ‘Upenamide (1): colorless solid, 2.2 mg (0.00037% based on wet weight); [R]D -9.44° (c 2.34, MeOH); for 1H and 13C NMR data, see Table 1 (assignments were made by interpretation of COSY, HMQC, and HMBC data); HRFABMS (oxalic acid:thioglycerol:glycerol) m/z obsd 523.3538 [M + H]+ (C32H46-
N2O4, ∆ 0.5 ppm); IR (thin film) υmax 3411, 2928, 1676, 1630, 1453, 1370, 1351, 1201, 1131, 970, 788 cm-1. Cytotoxicity Testing. Cytotoxicity assays were carried out by Instituto Biomar, S. A., Madrid, Spain.
Acknowledgment. We thank NSF, the Sea Grant College Program, Instituto Biomar, S. A., and PharmaMar, S. A. for financial support. Supporting Information Available: 1H, 13C, COSY, NOESY, HMQC, and HMBC NMR spectra for ‘upenamide are available.This material is available free of charge via the Internet at http/::pubs.acs.org. JO000789W
